The cellular behavior responsible for the development, maintenance and repair of differentiated cells and tissues is regulated, in large part, by intercellular signals conveyed via growth factors and other ligands and their receptors. The receptors for these intracellular signaling molecules are located on the cell surface of responding cells. Growth factors and other ligands bind to the receptors, thereby causing transduction of a signal across the cell membrane. Such signal transduction can occur by many modes, including pore formation and phosphorylation. Phosphorylation of tyrosines on proteins by tyrosine kinases is one of the key modes by which signals are transduced across the cell membrane. Indeed, several currently known protein tyrosine kinase genes encode transmembrane receptors for polypeptide growth factors and hormones.
Angiogenesis is generally thought to be heavily regulated by growth factors and other ligands. Angiogenesis, and the concurrent tissue development and regeneration, depends on the tightly controlled processes of endothelial cell proliferation, migration, differentiation and survival. Both stimulator and inhibitor ligands appear to interact, directly or indirectly, with cellular receptors during these processes. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane. Angiogenic stimulators induce endothelial cells to migrate through the eroded basement membrane. The migrating cells then form a xe2x80x9csproutxe2x80x9d off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel.
The ligands and receptors involved in endothelial cell regulation are beginning to be elucidated. In particular, endothelial growth factor receptors and their kinases have been discovered. For example, a gene encoding an endothelial cell transmembrane tyrosine kinase was described by Partanen et al. (Proc. Natl. Acad. Sci. USA 87:8913-17 (1990)). This gene and its encoded protein are called xe2x80x9cTIE,xe2x80x9d which is an abbreviation for xe2x80x9ctyrosine kinase with Ig and EGF homology domains.xe2x80x9d (See Partanen et al., Mol. Cell. Biol. 12:1698-1707 (1992); International Patent Publication WO 99/15653.) Enhanced TIE expression was shown during neovascularization to be associated with developing ovarian follicles and granulation tissue in skin wounds. (See Korhonen et al., Blood 80:2548-2555 (1992).) Thus, TIE protein is likely to play a role in angiogenesis.
Two structurally related rat TIE receptor-like tyrosine kinases, TIE-1 and TIE-2, have been reported; these receptors are encoded by distinct genes. (See Maisonpierre et al., Oncogene 8:1631-7 (1993).) Both genes were found to be widely expressed in endothelial cells of embryonic and postnatal tissues. Significant levels of TIE-2 transcripts were also present in other embryonic cell populations, including lens epithelium, heart epicardium and regions of mesenchyme. (See Maisonpierre et. al., supra.) The predominant expression of TIE receptors in vascular endothelia suggests that TIE plays a role in the development and maintenance of the vascular system, and in particular, angiogenesis.
Ligands of the TIE receptors have also been characterized. Two TIE-2 binding ligands, angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), have been identified. Ang-1 polypeptide interacts with the TIE-2 receptor tyrosine kinase. (See Maisonpierre et al., Science 277:55-60 (1997).) Ang-2 polypeptide is antagonistic to Ang-1 polypeptide, preventing binding of the activating ligand and blocking its ability to stimulate TIE-2 kinase activity and autophosphorylation. Ang-1 and Ang-2 do not bind TIE-1, however. Ang-1 and Ang-2 are about 60% identical; the amino acid sequences of these polypeptides share similar domain structure with an N-terminal coiled-coil region and a C-terminal fibrinogen-like domain. Northern (RNA) analysis shows that ANG-1 RNA is quite widely expressed, but that the expression of ANG-2 RNA is very limited. ANG-2 RNA is present only in tissues such as ovary, uterus, and placenta, which undergo vascular remodeling. Ang-1 is thought to be the same as the human TIE receptor ligand xe2x80x9chtie-2xe2x80x9d or xe2x80x9chTL-1.xe2x80x9d (See International Patent Publication WO 99/15653.)
Recently, other ligands for the TIE-2 receptor were identified. (See Valenzuela et al., Proc. Natl. Acad. Sci. USA 96:1904-09 (1999).) These ligands are called TIE ligand-3 (or angiopoietin-3 (Ang-3)) and TIE ligand-4 (or angiopoietin-4 (Ang-4)). Ang-3, a mouse polypeptide, appears to be antagonistic to Tie2 receptor while Ang-4, a human polypeptide, appears to be an agonist. The precise physiological role of Ang-3 and Ang-4 polypeptides remains to be elucidated.
Other TIE ligand homologues from humans, NL1 to NL6 and NL8, have also been identified. (See International Patent Publications WO 99/15653 and WO 99/15654.) One of these homologues, NL6, was identified by screening a cDNA library for sequences that encode secretory signals. Subsequent analysis of the full length NL6 cDNA revealed homology to TIE ligand receptors. The other homologues, NL1-5 and NL8, were identified by screening an EST database for sequences showing similarity to NL6. Based on their similarity to NL6, NL1-5 and NL8, were also proposed to be involved in angiogenesis. NL1 and NL8 were found to be capable of making cells tumorigenic. (See International Patent Publication WO 99/15653.)
The number of TIE ligand homologues, including Ang-1 to Ang-4 and the NL family, suggests that these ligands play diverse roles in angiogenesis. Further characterization of these ligands is, therefore an important step in understanding their roles in angiogenesis. In particular, persistent, unregulated angiogenesis occurs in a multiplicity of disease states, including tumor metastasis and abnormal growth by endothelial cells, and supports the pathological damage seen in these conditions. Thus, characterization of angiogenic factors may also facilitate the development of treatments for diseases related to (and hypothesized as being related to) angiogenesis. For example, tumor formation has been proposed to be dependent on angiogenesis. Thus, TIE ligand homologues that inhibit angiogenesis may provide therapeutic treatments for such tumors.
The present invention provides methods for modulating angiogenesis using anti-angiogenic Ang-7 polypeptides. The present invention further encompasses the use of Ang-7 polypeptides for the treatment of a disease or clinical condition where angiogenesis is relevant to the causation or treatment of the disease or clinical condition. In one embodiment, such diseases or conditions include, but are not limited to, cancer, wound healing, tumor formation, diabetic retinopathies, macular degeneration, cardiovascular diseases, and the like. Further uses of the Ang-7 polypeptides include treatment of clinical conditions involving angiogenesis in the reproductive system, including regulation of placental vascularization or use as an abortifacient. The present invention also encompasses pharmaceutical compositions containing the Ang-7 polypeptide and the use of such pharmaceutical compositions for the treatment of the above-mentioned diseases or clinical conditions.
One aspect of the present invention relates to the use of Ang-7 polypeptides having the amino acid sequence of SEQ ID NO:2, as well as biologically active or diagnostically or therapeutically useful fragments, variants, derivatives and analogs thereof. An additional aspect relates to the use of antibodies against the Ang-7 polypeptides of the present invention, especially antibodies which bind specifically to an epitope of the sequence described in SEQ ID NO:2, or a sequence that shares at least 60%, preferably at least 70%, more preferably at least 80%, still more preferably at least 90%, or most preferably at least 95% sequence identity over at least 20, preferably at least 30, more preferably at least 40, still more preferably at least 50, or most preferably at least 100 residues, to SEQ ID NO:2.
Another aspect of the present invention relates to the use of isolated ANG-7 nucleic acids encoding the Ang-7 polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA, as well as ANG-7 antisense nucleic acids. Such nucleic acids include the ANG-7 cDNA sequence having the nucleotide sequence of SEQ ID NO: 1. Another aspect relates to ANG-7 sequence fragments or variants that encode biologically active or diagnostically or therapeutically useful polypeptides. Such fragments or variants include sequences having all possible codon choices for the same amino acid or conservative amino acid substitutions thereof, such as the nucleotide sequence identified as NL1 in International Patent Publication WO 99/15653 (SEQ ID NO: 1), the disclosure of which is incorporated in its entirety by reference herein. Other variants include those nucleic acids that are capable of selectively hybridizing to a human ANG-7 cDNA (e.g., SEQ ID NO:1) under stringent hybridization conditions. Another aspect of the present invention relates to nucleic acid probes comprising polynucleotides of sufficient length to selectively hybridize to a polynucleotide encoding an Ang-7 polypeptide of the present invention.
Still another aspect of the present invention relates to processes for producing Ang-7 polypeptides, or biologically active and diagnostically or therapeutically useful fragments or variants thereof, by recombinant techniques through the use of recombinant vectors. A further aspect of the present invention relates to recombinant prokaryotic and/or eukaryotic host cells comprising an ANG-7 nucleic acid sequence encoding an Ang-7 polypeptide, or biologically active or diagnostically or therapeutically useful fragments or variants thereof. In a related aspect, nucleic acid constructs are provided that express ANG-7 nucleic acids and/or Ang-7 polypeptides, fragments or variants. Such constructs typically include a transcriptional promoter and a transcriptional terminator, each operably linked for expression of the ANG-7 nucleic acid or fragment thereof.
Another aspect of the present invention relates to processes involving expression of the polypeptides, or polynucleotides encoding the polypeptides, of the present invention for purposes of gene therapy. As used herein, gene therapy is defined as the process of providing for the expression of nucleic acid sequences of exogenous origin in an individual for the treatment of a disease condition within that individual.
A further aspect of the present invention relates to processes for utilizing Ang-7 polypeptides fragments, variants, derivatives, or analogs thereof, or ANG-7 polynucleotides or fragments, variants or derivatives thereof, for therapeutic purposes involving the modulation of angiogenesis, or the modulation of diseases or conditions in which angiogenesis is relevant to the disease or condition. Such diseases or conditions include, for example, the treatment of cancer, wound healing, diabetic retinopathies, macular degeneration, cardiovascular diseases, and clinical conditions involving angiogenesis in the reproductive system, including regulation of placental vascularization or use as an abortifacient. Such treatments further include the use of the Ang-7 polypeptides in protein replacement therapy and protein mimetics.
Another aspect of the present invention relates to diagnostic assays for detecting diseases or clinical conditions, or the susceptibility to diseases or clinical conditions, related to mutations in an ANG-7 nucleic acid sequence of the present invention and for detecting over-expression or underexpression of Ang-7 polypeptides encoded by such sequences.
These and other aspects of the invention will become evident upon reference to the following description and drawings.